Springer Semin Irnmunopathol (1999) 21 : 147-160 Springer Seminars in Immunopathology © Springer-Verlag 1999

The immunobiology of Acanthamoeba keratitis

Jerry Y. Niederkorn, Hassan Alizadeh, Henry F. Leher, James P. McCulley Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9057, USA

Background

Although only a few centimeters in diameter, the eye is composed of virtually every type of tissue found in the remainder of the body, as well as cellular and noncellular el- ements found nowhere else [37]. The eye is an extension of the brain, and, as such, conducts an enormous array of neurological functions. The million ganglion cells of the retina transmit 500 electrical signals per second, which in computer terms is equiv- alent to 1.5 × ]0 9 bits of information [37]. However, if the cornea loses transparency, complex functions of the retinal elements are rendered meaningless, and acute vision is preempted. To prevent this loss from happening, the eye utilizes anatomical, physi- ological, and immunological barriers to shield the cornea from injury and infection [43]. In many animals the eye is recessed in cranial sockets and surrounded by bony protuberances protecting it from blunt trauma. The eyelids and tear film serve as effective barriers against environmental agents, including air- and water-borne patho- gens. The cornea is exposed continuously to the external environment and potential patho- gens. One might expect any infectious agent encountered at the ocular surface would be met with a vigorous immune response that would promptly eliminate the pathogen and protect the cornea. However, in certain cases, a zealous immune response to corneal pathogens can have the opposite effect, contributing instead to loss of corneal transparency and blindness. Ironically, the three most common causes of infectious blindness - trachoma, herpes simplex virus (HSV) keratitis, and onchocerciasis - are immune-mediated diseases [43, 51, 65]. A recent study suggests the pathogenesis of one of the most common bacterial infections of the cornea, Pseudomonas aeruginosa keratitis, is also immune-mediated [26]. Thus, immune-mediated diseases of the cornea are not restricted to a specific category of pathogen, as the previous examples included a helminth (Onchocerca volvulus), a virus (HSV), an intracellular bacterium (Chlamydia trachomatis), and an extracellular bacterium (P. aeruginosa). With this

Correspondence to: J. Y. Niederkom 148 J.Y. Niederkorn et al. information in mind, we suspected the pathogenesis of another corneal infection, Acanthamoeba keratitis, also might be immune-mediated. Acanthamoeba keratitis is a sight-threatening corneal disease caused by patho- genic, free-living amoebae [3-5]. At least eight species of Acanthamoeba have been implicated in corneal infections: A. castellanii, A. culbertsoni, A. polyphaga, A. hatch- etti, A. fhysodes, A. lugdunesis, A. quina, and A. griffini [59]. Acanthamoeba spp. are ubiquitous organisms that can be isolated from a wide range of environmental sites, in- cluding fresh water reservoirs, salt water, swimming pools, hot tubs, ventilation ducts, soil, bottled water, and even eyewash stations [9, 31, 33, 34, 50, 55, 56, 58]. Exposure to Acanthamoeba spp. is apparently common, as 50-100% of the normal population possesses circulating antibodies specific for Acanthamoeba antigens [10, 11]. More- over, viable Acanthamoeba trophozoites can be isolated from the noses and throats of asymptomatic individuals [57]. The first case of Acanthamoeba keratitis was reported in 1973 by Jones et al. [24]. Between 1973 and 1981, only five additional cases were reported. The number of cases increased gradually from 1981 to 1984, with an increased incidence of Acan- thamoeba keratitis occurring during the late 1980s [42, 61]. Although Acanthamoeba keratitis is no longer reported to the Center for Disease Control, the general impression among clinicians is that the incidence is decreasing in North America but not in the United Kingdom.

Clinical features

Acanthamoeba keratitis is closely associated with contact lens wear, which appears to be the leading risk factor [40]. Over 80% of the patients diagnosed with Acan- thamoeba keratitis wear contact lenses, and soft contact lenses account for approxi- mately 75% of the cases [42]. One of the most curious features ofAcanthamoeba ker- atitis is the exquisite pain, which is not commensurate with the clinical signs. Radial neuritis or trophozoite infiltration of the corneal nerves is pathognomonic for Acan- thamoeba keratitis [41]. The severe pain and involvement of the corneal nerves by trophozoites may be related to the behavior of the trophozoites, which demonstrate a strong chemotactic response to cells of neural crest origin [54]. In vitro studies have shown that, although Acanthamoeba trophozoites kill corneal cells, they display an even greater propensity to lyse cells of neural origin [53, 54]. In addition to intense oc- ular pain, the early features of corneal infection with Acanthamoeba spp. include eye- lid-reactive ptosis, conjunctival hyperemia, and lack of discharge. Chemosis and nod- ules appear in the later stages of the disease. Corneal epithelium and sometimes the stroma are affected in the early stage of infection. As the disease progresses, stromal involvement becomes more pronounced with the development of stromal infiltrates and characteristic ring infiltrates. Other later symptoms of Acanthamoeba keratitis are the occurrence of lacuna-like changes in the ring, satellite lesions, necrotizing inflam- mation, and stromal abscess formation.

Pathogenesis

The pathogenesis of Acanthamoeba keratitis can follow two pathways [16, 64]. The first pathway is restricted to the epithelium without involvement of the stroma and has Immunobiologyof Acanthamoeba keratitis 149 a good prognosis. The second pathway culminates in the parasites entering the stroma, where they produce extensive necrosis and edema and provoke intense inflammation [16]. The first step in the pathogenesis of Acanthamoeba keratitis is the binding of trophozoites to the corneal epithelial surface. Animal studies have shown that binding of Acanthamoeba trophozoites to corneal epithelial cells and corneal buttons in vitro correlates with pathogenicity in vivo [20, 44, 69]. That is, Acanthamoeba trophozoites bind poorly to the corneas of animal species resistant to experimental corneal infec- tion, but adhere extensively to human, pig, and Chinese hamster corneas [44]. Acan- thamoeba trophozoites express a mannose-binding receptor, which facilitates adhesion of the parasites to mannosylated proteins on corneal epithelial cells [74]. Panjwani et al. [48] have demonstrated that free mannose strongly inhibits the binding of Acan- thamoeba trophozoites to the corneal epithelium and invasion of corneal buttons in vitro [48]. The presence of free mannose blocks parasite-mediated cytolysis of corneal cells in short-term in vitro assays [49]. However, trophozoites exposed to free man- nose 48 h or longer are induced to release one or more soluble cytolytic factors, which mediate contact-independent cytolysis of corneal epithelial cells in vitro [30]. Binding of Acanthamoeba trophozoites to either free mannose or mannosylated proteins on corneal cells promotes contact-dependent and contact-independent cytolysis of corneal cells. Pathogenic amoebae elaborate a variety of cytolytic molecules that might be elicited by engagement of the mannose receptor. Hadas and Mazur [17] examined eight species of Acanthamoeba and detected a 35-kDa serine proteinase and a 65-kDa cysteine protease. Both of these proteinases, however, are significantly smaller than the serine protease secreted by mannose-treated A. castellanii trophozoites [30]. Enta- moeba histolytica produces 5-kDa and 14-kDa protein complexes, called amoeba- pores, which form ion channels leading to the osmotic lysis of eukaryotic cells [12]. The Acanthamoeba-derived cytolytic factor induced by mannose has a molecular mass in excess of 100 kDa and is too large to be an amoebapore. In addition to killing target cells by disruption of the cell membrane, trophozoites also produce a significant amount of cell death by inducing apoptosis of the target cells [1, 53]. Following binding and erosion of the epithelial surface, trophozoites invade the deeper regions of the corneal epithelium and penetrate the stroma. Corneal invasion probably is facilitated by proteases secreted by the trophozoites [68]. A. castellanii elaborates a variety of proteases, including a 45- to 50-kDa plasminogen activator, termed Acanthamoeba plasminogen activator (aPA), which is detected in pathogenic strains of Acanthamoeba spp. but not found in non-pathogenic strains of A. castellanii [38]. It is possible, although yet to be proven, that a critical step in the pathogenic cas- cade of Acantharnoeba keratitis is the parasite's elaboration of plasminogen activator and the ensuing generation of plasmin, which in turn facilitates the parasite's invasion of the corneal epithelium and stroma. Stromal disease occurs late and is characterized by a ring infiltrate or abscess that can consist of single, multiple, or overlapping rings. The underlying cause of the ring infiltrates is unclear. Some have suggested it is the result of infiltrating neutrophils and their release of proteolytic enzymes that degrade the collagen matrix of the stroma [16]. Other investigators have proposed that the ring infiltrates are products of the or- ganisms, with only minimal contribution by host inflammatory cells [3-5]. We and others have reported that Acanthamoeba spp. secrete collagenolytic enzymes, which digest collagen shields and purified type I collagen in vitro [19, 39]. Acanthamoebae produce a variety of other proteases that might contribute to stromal melting [17]. Im- portantly, intrastromal injection of sterile culture medium from axenic cultures of 150 J.Y. Niederkornet al.

A. castellanii into the corneas of rats produced characteristic ring infiltrates and corneal lesions that clinically and histopathologically mimicked those found in patients diag- nosed with Acanthamoeba keratitis [19]. These findings suggest that parasite-derived collagenolytic enzymes contribute to the formation of the ring-infiltrates that are char- acteristic of Acanthamoeba keratitis.

Immune response to corneal infection with Acanthamoebae

Exposure to Acanthamoebae must be common as 50-100% of the normal population possess antibodies against Acanthamoeba antigens [10, 11]. Analysis of Acan- thamoeba-specific T cell proliferative responses in healthy, asymptomatic individuals revealed the peripheral blood lymphocytes of 50% of the human subjects demonstrated significant T cell proliferative responses to Acanthamoeba antigens [67]. Since none of the individuals in the aforementioned studies had a history of Acantharnoeba keratitis, it is assumed that the observed immune responses to Acanthamoeba antigens were the result of environmental exposure to the organism. Whether corneal infection with Acanthamoeba spp. provokes systemic immune responses in humans remains unknown. Animal studies have shown that corneal infection with A. castellanii failed to induce either delayed-type hypersensitivity (DTH) or serum IgG antibody against parasite antigens [2, 46, 71 ]. Failure to induce cell-mediated and humoral immunity did not re- sult in anergy or tolerance, since subsequent intramuscular immunization with parasite antigens elicited robust DTH and IgG antibody responses [71 ]. The inability of corneal infections to induce primary cell-mediated immune responses was due to the absence of resident antigen-presenting cells in the central cornea because induction of Langer- hans cell (LC) migration into the central cornea prior to infection with Acanthamoeba promoted the development of parasite-specific DTH. Although the presence of resi- dent LC did not promote the development of a primary humoral immune response, subsequent intramuscular immunizations elicited heightened parasite-specific IgG an- tibody production indicative of an anamnestic response [71]. These findings suggest that in the absence of resident antigen-presenting cells, corneal infection with Acan- thamoeba fails to stimulate primary cell-mediated immunity, but does partially acti- vate the B cell arm of the immune apparatus. It is interesting that the induction of pe- ripheral LC into the central corneal epithelium prior to corneal infection promotes the development of parasite-specific DTH, but does not exacerbate corneal disease. The role of specific immune effector mechanisms in the resistance to Acan- thamoeba keratitis is confusing. Histopathological investigations of human and animal specimens revealed that severe inflammatory responses are characteristic of Acan- tharnoeba keratitis [28, 35]. Macrophages are predominant during the early stage of experimental infection [28]. Histopathological investigations of late stage disease in human subjects revealed the presence of macrophages and neutrophils, but only occa- sional lymphocytes [16, 25, 35]. In vitro studies demonstrated both neutrophils and macrophages are capable of killing Acanthamoeba trophozoites [13, 63]. Parasite-specific antibody and interferon- 3t augment macrophage-mediated killing in vitro (Fig. 1). The role of neutrophils in protecting against corneal infection with Acanthamoeba spp. is unclear. However, an- imal studies suggest macrophages provide a significant level of protection against in- fection [70]. In situ depletion of conjunctival macrophages with the macrophagicidal drug dichloromethylene diphosphonate (clodronate) profoundly exacerbated the sever- Immunobiologyof Acanthamoeba keratitis 151

MAC + IMMUNE SERUM

IMMUNE SERUM w/o MAC

MAC + NORMAL SERUM

MAC + INTERFERON

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% KILLING OF TROPHOZOITES Fig. L Macrophage-mediatedkilling of Acanthamoeba castel/anii trophozoites. Rat peritoneal macrophages (MAC) were armed with either hyperimmune anti-Acanthamoeba antiserum, normal rat serum, or murine interferon-y prior to co-incubation with radiolabeled trophozoites for 6 b in vitro. Percent killing was deter- mined by isotope release. The details of this experiment have been published previously [63]

ity, chronicity, and incidence ofAcanthamoeba keratitis in Chinese hamsters (Figs. 2, 3). The most likely explanation to account for the exacerbation of Acantharnoeba keratitis in animals treated with clodronate is that macrophages act as a first line of defense and eliminate the bulk of the Acanthamoeba trophozoites early in the infec- tious process. This defense would prevent the tropbozoites from becoming widely disseminated. The hypothesis that the macrophage acts as a first line of defense is supported by in vitro studies showing that macrophages demonstrate a strong chemo- tactic response to Acanthamoebae and can directly kill trophozoites in vitro [63], The paucity of macrophages in corneal biopsy specimens and corneal buttons from Acan- thamoeba keratitis patients who received penetrating keratoplasty calls into question the role of macrophages in controlling Acanthamoeba infections of humans [25]. Yet most histopathological studies on Acanthamoeba keratitis patients have examined corneal specimens during the latter stages of disease and not during the acute phase [16, 25, 35]. We suspect if macrophages serve as important barriers to corneal infec- tion, they would exert their effect by preventing the initiation of infection and the ap- pearance of clinical signs. This would explain the absence of macrophages in previ- ous histopathological studies on human corneal specimens [25] and their presence in experimental animals [6, 27, 28]. Also, the present results, as well as previous find- ings, indicate that the normal conjunctival macrophage population does not totally inhibit corneal infection in all animals, but appears to limit the severity and chronic- ity of corneal disease. This effect probably is exerted just after the parasites bind to 152 J.Y. Niederkom et al.

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DAYS POST INFECTION Fig. 2. Depletion of conjunctival macrophages increases the susceptibility of Chinese hamsters to Acan- tharnoeba keratitis and prevents resolution of corneal disease. Liposomes containing either dichloromethyl- ene diphosphonate (clodronate) or phosphate-buffered saline (control) were injected into the conjunctivae of Chinese hamsters prior to the application of Acanthamoeba-laden contact lenses. These experiments have been described elsewhere [70]

the corneal epithelial surface and would not be detected readily by conventional histopathological methods. Although a high percentage of the normal population possesses circulating anti- bodies that react with Acantharnoeba antigens, little evidence suggests serum antibod- ies, namely IgG, affect the course of Acanthamoeba keratitis in human subjects. In both pig and Chinese hamster models ofAcanthamoeba keratitis, high titers of IgG an- tibodies against Acanthamoeba antigens can be induced by intramuscular immuniza- tion, yet the presence of circulating anti-Acanthamoeba IgG antibodies fails to affect the incidence, severity, chronicity, or clinical features of Acanthamoeba keratitis [2, 46]. These observations are especially puzzling considering the susceptibility of Acan- thamoeba trophozoites to complement-dependent lysis by antibody in vitro [13]. Acanthamoeba trophozoites readily activate the complement cascade via the alterna- tive pathway and succumb to complement-mediated lysis in vitro, even in the absence of antibody [14]. The pathogenicity of other pathogenic/free-living amoebae such as Naegleria spp. is correlated with the trophozoites' resistance to complement-mediated lysis [73]. There appears to be no such correlation with Acanthamoeba spp.; however, because trophozoites capable of activating the complement cascade via the alternative pathway are highly pathogenic in experimental animals, even in the face of circulating anti-Acantharnoeba antibodies and an intact complement system [2, 46], the capacity of trophozoites to evade complement-mediated injury in the cornea is enigmatic. It is Immunobiologyof Acanthamoeba keratitis 153

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DAYS POST INFECTION Fig.3. Depletion of conjuncdval macrophages exacerbates Acanthamoeba keratitis in Chinese hamsters. Conjunctival macrophages were depleted with clodronate-containing Iiposomes as described in Fig. 2 and elsewhere [70]. "Severity of Infection" is a composite of clinical scores, which scored corneal edema, opac- ity, ulceration, neovascularization, and stromal infiltration as described previously [70]. Scores from two to three independent observers were averaged possible that complement regulatory proteins, such as decay-accelerating factor (CD55), which are expressed on the corneal epithelium and in the tears, are able to in- activate complement locally and thereby protect the parasite from the lytic stage of the complement cascade [8, 29].

Do Langerhans cells affect Acanthamoeba keratitis?

The absence of r~sident antigen-presenting LC in the central corneal epithelium pro- foundly affects the immunobiology of the cornea and corneal allografts [45, 47]. LC are bone marrow-derived dendritic antigen-presenting cells, which present both ex- ogenous and endogenous antigens to T cells [62]. LC are potent inducers of Thl im- mune responses like DTH [62]. Unlike other epithelial surfaces such as the skin; the central corneal epithelium is normally devoid of LC [23]. However, a variety of stim- uli elicit swift centripetal migration of LC from the limbus into the central regions of the cornea [23]. These include infections, phagocytic stimuli, minor surgical proce- dures, or injection of interleukin-1 (IL-1). The paucity of LC in the central corneal ep- ithelium appears to be an adaptation for reducing the likelihood of eliciting DTH re- sponses to antigens encountered in the central cornea. Blunting the induction and ex- pression of DTH would be important for maintaining normal corneal function and clar- 154 J.Y. Niederkorn et al. ity, as DTH reactions characteristically inflict significant nonspecific damage to nor- mal tissues. Compelling evidence suggests that the leading cause of infectious blindness in North America, HSV stromal keratitis (HSK), and in developing countries, trachoma, are DTH-mediated disease [43, 65]. Thl responses play an important role in the patho- genesis of corneal infections with P. aeruginosa infegted mice [26]. Using an animal model of HSK, Hendricks et al. [21] showed the severity of HSV keratitis correlated with the density of LC in the central corneal epithelium. Corneal lesions were most se- vere in eyes pretreated with IL- 1 to induce centripetal migration of peripheral LC into the central corneal epithelium prior to exposure to topical infection with HSV [21]. DTH and keratitis were dramatically reduced if LC were depleted by ultraviolet irra- diation prior to exposure to HSV [21]. Based on these findings, we suspected that repeated exposure to Acanthamoeba spp. would result in systemic immunization and that subsequent ocular exposure would provoke a DTH response in the cornea, resulting in immune-mediated keratitis. The rationale for this hypothesis was further supported by the observation that Acan- thamoeba spp. are found in a wide variety of environmental sites, thereby promoting frequent exposure to Acanthamoeba antigens. The high frequency of seropositivity in the normal population of asymptomatic individuals is consistent with this prediction [10, 1 i]. To our surprise, inducing centripetal migration of peripheral LC into the cen- tral cornea - either by intracorneal injection of IL-1 or by instillation of sterile latex beads - prior to infection with Acanthamoeba trophozoites not only failed to exacer- bate disease, but, instead, produced a remarkable resistance to infection [69]. The pres- ence of LC in the central corneal epithelium profoundly affected the incidence and severity of Acanthamoeba keratitis. Clinical signs of keratitis developed in only 14% of the animals pretreated with latex beads compared to a 60% incidence of infection in control animals. In the few latex bead-treated animals that did develop Acanthamoeba keratitis, all clinical parameters were reduced by > 50% compared to controls [69]. The mechanism whereby induction of LC migration into the central cornea protects against Acanthamoeba keratitis remains a mystery. As mentioned earlier, induction of LC migration into the central cornea prior to ocular challenge with Acanthamoeba trophozoites results in the induction of robust Thl immunity against the parasite anti- gens [71]. DTH does not appear to play a role in protecting against corneal challenge, however, because pigs and Chinese hamsters immunized intramuscularly prior to ocu- lar challenge develop strong DTH immunity to parasite antigens, yet show no evidence of protection against corneal challenge [2, 46, 71]. Chinese hamsters treated by either latex bead instillation or intracorneal injection with IL-1 demonstrate resistance to in- fection within 2-3 days - a time frame which is too swift for the generation of a con- ventional immune response. This protective effect is not due to impairment of the par- asite's capacity to attach to the corneal epithelium, since neither the latex bead nor IL- l treatment affects the binding of the trophozoites to the corneal buttons in vitro [69]. The simplest explanation to account for the protective effect of latex bead or IL-1 treatment is that LC directly lyse trophozoites or induce them to encyst. However, in vitro studies using cutaneous LC and A. castellanii trophozoites have failed to provide any evidence that LC can kill or induce the encystment of Acanthamoeba trophozoites (unpublished findings). These observations have been recreated nunaerous times in our laboratory by different investigators, yet we are unable to determine the underlying mechanism. Unraveling this mystery may provide important insights into the biology and immunology of Acanthamoeba keratitis, and perhaps other corneal infections as well. Immunobiology of Acanthamoeba keratitis 155

Oral immunization against Acanthamoeba keratitis

The ocular mucosal epithelium is exposed regularly to the environment, but is pro- tected against pathogens by a variety of humoral components present in the tears in- cluding lactoferrin, lysozyme, and lactoperoxidase. In addition to innate humoral de- fense mechanisms, the tears cOntain IgA, IgG, and IgM immunoglobulins [ 15, 18]. Yet IgA is clearly the most abundant immunoglobulin in mammalian tears [ 15, 18, 72]. In rodents, tear IgA is produced by plasma cells in the lacrimal glands and is not derived by transudation from the serum [52, 66]. Immunization via mucosal surfaces is known to elicit the production of secretory IgA antibody in multiple mucosal secretions and is the basis for the concept of the common mucosal immune system. Cholera toxin is a potent mucosal adjuvant, which enhances antigen uptake by Peyer's patches and po- tentiates the IgA antibody response to antigens delivered via mucosal surfaces [7, 22, 32]. Using cholera toxin as an adjuvant, we have shown that oral immunization with Acanthamoeba antigens induces strong resistance to corneal infections with A. castel- lanii in both the Chinese hamster and pig models ofAcanthamoeba keratitis (Table 1). Protection against ocular infections correlated with the appearance of parasite-specific IgA antibodies in mucosal secretions including the tears (manuscript submitted for publication). In vitro studies confirmed that anti-Acanthamoeba IgA antibodies effec- tively inhibited the binding of trophozoites to corneal epithelial cells, but did not affect parasite viability (Fig. 4). This confirmation is consistent with subsequent studies in the pig model, which demonstrated that oral immunization failed to mitigate the severity or hasten the resolution of Acanthamoeba keratitis if immunization was initiated after corneal infection was established (manuscript submitted for publication). Although such hosts developed high tear IgA antibody titers, they exhibited corneal infections indistinguishable from immunologically naive hosts. When the animals were subse- quently rechallenged, they showed solid resistance to ocular infection with Acan- thamoeba (manuscript submitted for publication). Based on these findings, we favor the hypothesis that oral immunization elicits the production of IgA antibodies, which

Table 1. Route of immunization affects resistance to Acanthamoeba keratitis

Route of Host Immune response Incidence immunization~ of DTH IgG IgA infection Reference

None Chinese hamster - - 71% [47] and Leher et al. b Acanthamoeba Ag oral Chinese hamster ND ND + 20% Leher et al. b Acanthamoeba Ag IM Chinese hamster + + ND 75% [47] None Pig ND - 100% [45, 46] and Leher et a15 Acanthamoeba Ag oral Pig ND + + 0% [45, 46] and Leher et al£ Acanthamoeba Ag IM Pig + + ND 100% [45, 46]

ND, Not determined ~'Acanthamoeba Ag = aqueous parasite antigen given either orally or intramuscularly (IM) along with cholera toxin adjuvant b Leber HE, Alizadeh H, Taylor WM, Shea AS, Silvany RS, van Klink F, Jager MJ, Niederkorn JY (1998) Tear IgA protects against ocular infection with Acanthamoeba by inhibiting parasite adhesion (submitted for publication) c Leber H, Kinoshita K, Alizadeh H, Zaragosa E He YG, Niederkom JY (1998) Specific tear IgA prevents the induction of Acanthamoeba keratitis but fails to affect ongoing disease (submitted for publication) 156 J.Y. Niederkorn et al.

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Conclusions

Recrudescence is a troubling and puzzling feature of Acanthamoeba keratitis, suggest- ing ocular infection fails to arouse the systemic immune apparatus. Results from ani- mal studies support this proposition and suggest that the conspicuous absence of resi- dent antigen-presenting cells in the corneal epithelium creates an immunological "blind spot," which prevents the immune system from perceiving the presence of the parasite within the corneal milieu. The inability of corneal infections with Acan- thamoeba spp. to elicit perceptible cell-mediated and humoral immune responses rules out a role for immune-mediated mechanisms in the pathogenesis of Acanthamoeba keratitis. Even when the systemic immune apparatus is activated, however, it only ap- pears to be capable of preventing the initiation of infection and is impotent in affecting ongoing corneal disease. The latter observation is paradoxical. Acanthamoeba tropho- Immunobiology of Acanthamoeba keratitis 157 zoites are highly susceptible to cellular and humoral immune effector elements in vitro, yet the presence of IgG antibody and DTH responsiveness to Acanthamoeba antigens does not seem to affect the incidence or severity of Acanthamoeba keratitis in experimental animals. The remarkable immunity provided by secretory IgA antibody in the tears of orally immunized Chinese hamsters and pigs indicates at least one com- ponent of the immunological apparatus is capable of preventing corneal infection with Acanthamoeba. Perhaps this should come as no surprise, as the tear film is the first and most fundamental barrier to corneal infection, and IgA is the most abundant immuno- globulin produced in the body. In fact, more IgA is produced each day than all of the other immunoglobulin isotypes combined [36, 60]. The immune system's significant investment in IgA production is a testament to its value as a defensive modality.

Acknowledgement This work supported by NIH grant EY09756, and an unrestricted grant from Research to Prevent Blindness, Inc., New York, NY.

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